During development, the lung mesenchyme gives rise to non-epithelial structures, while in the mature lung the mesenchyme responds to perturbations to maintain homeostasis. The function, or malfunction, of mesenchymal cells (e.g., fibroblasts, myofibroblasts and smooth muscle cells) is intimately involved with numerous lung diseases, including emphysema, asthma, fibrosis, pulmonary hypertension, cancer and bronchopulmonary dysplasia. There are significant gaps in our knowledge of how resident mesenchymal cells function over time in the lung. Major challenges include the need for additional markers that delineate functionally distinct subtypes of mesenchymal cells, the precise identification of mesenchymal cells that function as resident progenitor or stem cells, and the identification of new signaling pathways or other molecules in mesenchymal cells that can be therapeutically targeted. The broad goal of our work is to identify novel markers of fibroblast subsets, in particular stem/progenitor cells that might be used to identify and therapeutically target these cells in disease states. Sonic hedgehog (Shh) is a signaling molecule involved in mesenchyme proliferation and differentiation during lung development. In lung, Shh is produced by epithelium and induces critical responses in mesenchyme required for branching morphogenesis. While Shh production largely ceases by birth, we find there remains a population of mesenchymal cells in adult lung responding to Shh or a similar Hedgehog (Hh) ligand. These cells are identifiable in Gli1-lacZ reporter mice (the Gli1 gene is both a component of the Hh signaling pathway and a transcriptional target of Hh signaling). The Hh-responding cells have a distinctive location in the adventitia of airways and pulmonary vessels, and in the visceral pleura. By colocalization studies we find that the cells are largely col1+SMA- fibroblasts. Based on our data and recent results on mesenchymal stem/progenitor cells from other groups, we hypothesize that the Gli1-lacZ+ cells are an important population of mesenchymal cells with progenitor/stem cell capability. To test this hypothesis we propose two broad aims: (1) to genetically fate-map the Hh-responding cells under various conditions to determine their ability to expand and contribute to different cell lineages in the lung, and (2) to develop techniques to isolate and culture these cells, and characterize their behavior in vitro. The project will be accomplished by careful analysis of various Hh-reporter mouse strains and cells derived from these mice, and should have broad implications for lung biology and disease. PUBLIC HEALTH RELEVANCE: Lung mesenchymal cells are responsible for synthesis of collagen and other structural proteins, the contractile activity of vessels and airways, repair processes after injury, and maladaptive scarring. Although mesenchymal cells are thought to exist in multiple subtypes that differ in their functions, including their capacity for differentiation, renewal and stem cell behavior, and these functions are critical in diseases such as asthma, pulmonary hypertension and lung fibrosis, our understanding of these subtypes remains inadequate, in part because of a lack of definitive markers by which to identify them. We found that a distinct subset of lung mesenchymal cells is identifiable by activation of the Hedgehog signaling pathway (a pathway that has been most studied in the context of embryonic development), and hypothesize that these cells have important proliferative and progenitor functions in the adult that are relevant to many disease states and that could be targeted for therapeutic benefit;in this application for an R21 award we propose to develop procedures and perform experiments to isolate and characterize these cells in vitro and in vivo.